This application presents novel methods of detection, diagnosis, prognosis, prevention, and treatment of inflammatory bowel disease (IBD) and cancers, specifically colorectal cancers (CRCs).
IBD, inclusive of ulcerative colitis (UC) and Crohn's disease (CD), is a chronic inflammatory disease of the gastrointestinal (GI) tract. At present, monoclonal antibodies against TNF-α remains one of the most effective treatments against IBD, in addition, aminosalicylates, corticosteroids and immunosuppressants are also used. However, due to the multi factorial nature of the disease, flare-ups of the disease and side effects associated with the different treatment approaches, in particular corticosteroids are common. Although both genetic and environmental factors contribute to IBD pathogenesis, epigenetic regulators, such as microRNAs may also play an important role in IBD.
CRC is cancer that starts in either the colon or the rectum. Although early intervention by surgery can cure up to 90% patients, CRC is often diagnosed at an advance stage. Colonoscopy remains one of the most sensitive CRC screening tests currently available. Genetic testing of stool DNA is under examination for the feasibility as screening tools. On the other hand, depending on the stages, CRC can be treated by either surgery alone or in combination with chemotherapy. Novel therapeutic modalities are still actively sought. Research by others has shown that AFTPH consists of binding sites with other proteins involved in endocytosis and is crucial to intracellular transport. Our evidence both in vitro (human cancer cell lines) and in vivo (mouse xenograph model) shows that reducing AFTPH levels promotes tumor growth. In addition, we have also shown that AFTPH levels decrease in colon cancer tissue samples when compared to colonic biopsies from normal subjects. Thus. AFTPH represents a novel candidate for CRC screening and a novel therapeutic target for CRC.
G protein-coupled receptor (GPCR) recycling allows cell resensitization to ligand stimulation and sustains signaling. Although microRNAs regulate many physiological functions, their involvement in GPCR recycling is unknown. We have reported that the neuropeptide neurotensin is involved in the pathophysiology of colon cancer and intestinal inflammation. We also showed that recycling of the GPCR neurotensin receptor 1 (NTR1) regulates proliferative and pro-inflammatory responses in colonocytes. Here, we show that in human colonocytes, NT increases miR-133α expression that enhances NTR1 recycling, but not endocytosis, through direct down-regulation of aftiphilin (AFTPH), a protein associated with trafficking. NTR1 induced miR-133α expression by reducing binding of Zinc finger E-box binding homeobox 1 (ZEB1) to the miR-133α promoter, MiR-133α-regulated NTR1 recycling was linked to NT-associated tumor formation. Increased miR-133α and decreased AFTPH mRNA expression was found in human colon cancers, while miR133α and AFTPH mRNA levels were correlated with tumor stage, Thus, we demonstrate a novel mechanism of a GPCR recycling through microRNA expression that may provide a new target for therapeutic approaches in colon cancer.
Neurotensin (NT) is a 13-amino add neuropeptide expressed in the central nervous system and the intestine (Polak, J. M., et al., 1977. Specific localisation of neurotensin to the N cell in human intestine by radioimmunoassay and immunocytochemistry. Nature 270:183-184; Castagliuolo, I., et al., 1999. Neurotensin is a proinflammatory neuropeptide in colonic inflammation. J Clin Invest 103:843-849). Its high affinity G protein-coupled receptor (GPCR) neurotensin receptor 1 (NTR1) (Tanaka, K.; et at., 1990. Structure and functional expression of the cloned rat neurotensin receptor. Neuron 4:847-854) is overexpressed in colon cancer cell lines (Bakirtzi, K., et 2011. Neurotensin Signaling Activates MicroRNAs-21 and -155 and Akt, Promotes Tumor Growth in Mice, and Is Increased in Human Colon Tumors. Gastroenterology 141:1749-1761.e1741) and intestinal tumors (Gui, X., et al., 2008. Increased neurotensin receptor-1 expression during progression of colonic adenocarcinoma. Peptides 29:1609-1615).
In the intestine, NTR1 signaling promotes both proliferation and inflammation through MAP kinase and NF-κB pathways (Castagliuolo, I., Wang, C. C., Valenick, L., Pasha, A., Nikulasson, S., Carraway, R. E., and Pothoulakis. C. 1999. Neurotensin is a proinflammatory neuropeptide in colonic inflammation. J Clin invest 103:843-849; Zhao, D., et al., 2004. Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells. J Biol Chem 279:43547-43554; Zhao, D., et al., 2007. Neurotensin stimulates expression of early growth response gene-1 and EGF receptor through MAP kinase activation in human colonic epithelial cells. Int J Cancer 120:1652-1656; Zhao, D., et al., 2001. Signal transduction pathways mediating neurotensin-stimulated interleukin-8 expression in human colonocytes. J Biol Chem 276:44464-44471). NTR1 activation induces differential expression of 38 microRNAs in human colonocytes overexpressing NTR1 (NCM460-NTR1) (Bakirtzi, K.; et al., 2011. Neurotensin Signaling Activates MicroRNAs-21 and -155 and Akt, Promotes Tumor Growth in Mice, and is increased in Human Colon Tumors. Gastroenterology 141:1749-1761.e1741).
MicroRNAs are short (19-25 nucleotides); single-stranded RNA molecules, acting as negative transcriptional or post-transcriptional regulators. They bind to the 3′ untranslated regions (UTRs) of transcripts (McKenna, et al., 2010. MicroRNAs control intestinal epithelial differentiation; architecture; and barrier function. Gastroenterology 139:1654-1664; 1664 e1651) and lead to messenger RNA (mRNA) degradation, or inhibition of translation into protein (Bartel, D. P. 2009. MicroRNAs: target recognition and regulatory functions. Cell 136:215-233). MicroRNAs regulate many physiological functions, including inflammation (Contreras. J., et al., 2012. MicroRNAs in inflammation and immune responses, Leukemia 26:404-413), metabolism (Rottiers, V., et al., 2012. MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol 13:239-250) and cancer development (Croce, C. M. 2009. Causes and consequences of microRNA dysregulation in cancer, Nat Rev Genet 10:704-714), including colon cancer (Schetter, A. J., et al., 2011. Alterations of microRNAs contribute to colon carcinogenesis. Semin Oncol 38:734-742).
NTR1 is a “class B” receptor with sustained and high affinity binding to β-arrestins, which control receptor desensitization and endocytosis (Oakley, R. H., et al., 2000. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J Biol Chem 275:17201-17210; Oakley, R. H., et al., 2001, Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-beta-arrestin complexes after receptor endocytosis. J Biol Chem 276:19452-19460). The activated NTR1 internalizes with β-arrestins in NCM460-NTR1 cells and recycles from Rab5a+ early endosomes in a endothelin-converting enzyme-1 (ECE-1)-dependent manner (Law. I. K. M., et al, 2012. Neurotensin-induced pro-inflammatory signaling in human colonocytes is regulated by beta-arrestins and endothelin-converting enzyme-dependent endocytosis and re-sensitization of NT receptor 1. Journal of Biological Chemistry).
GPCR trafficking regulates signaling since receptor interaction with β-arrestins mediates desensitization and endocytosis, and receptor recycling generally mediates resensitization (Schmidlin, F., et al., 2001. Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor, J Biol Chem 276:25427-25437; Roosterman, D., et al., 2007. Endothelin-converting enzyme 1 degrades neuropeptides in endosomes to control receptor recycling. Proc Natl Aced Sci USA 104:11838-11843).
When cells are continuously exposed to NT, NTR1 recycling and resensitization are required for sustained signaling (Law, I. K. M., et al., 2012. Neurotensin-induced pro-inflammatory signaling in human colonocytes is regulated by beta-arrestins and endothelin-converting enzyme-dependent endocytosis and re-sensitization of NT receptor 1. Journal of Biological Chemistry). Since differential microRNA expression in response to NT in human colonocytes (Bakirtzi, K., et al., 2011. Neurotensin Signaling Activates MicroRNAs-21 and -155 and Akt, Promotes Tumor Growth in Mice, and Is Increased in Human Colon Tumors. Gastroenterology 141:1749-1761,e1741) coincides with NTR1 internalization and recycling to the plasma membrane (Law, I. K. M., et al., 2012. Neurotensin-induced pro-inflammatory signaling in human colonocytes is regulated by beta-arrestins and endothelin-converting enzyme-dependent endocytosis and re-sensitization of NT receptor 1. Journal of Biological Chemistry), we hypothesized that some of the NT-regulated microRNAs may play a role in these processes.
Our results indicate that NT-induced miR-133α expression regulates NTR1 recycling to the plasma membrane. We show that aftiphilin (AFTPH) is a downstream target of miR-133α that regulates NTR1 recycling as well as colonic tumor growth in vitro and in vivo. This is the first study providing evidence for an important role of microRNAs in regulation of GPCR recycling that is linked to development of colon cancer.